* Protein ID (NP or XP #) or Wolbachia #: NP_465325.1 (use De La O's Oligos for ordering - Dr. B 090313)
* Organism: Listeria monocytogenes
Etiologic Risk Group (see link below): Risk Group 2 - Bacterial agents including Chlaymidia
* Background / Disease Information: The Most Deadly Born food pathogen. Induces muscle aches, fever and diarrhea. Main route of infection is through uncooked and unwashed food.
Essentiality of this protein: Protein required to induce host to engulf bacteria. Upon entry to the cytosol bacteria can reproduce. http://www.ncbi.nlm.nih.gov/pubmed/21444667
Structure Available (PDB or Homology model) http://www.rcsb.org/pdb /explore/explore.do?structureId=1YWF - QUERY Coverage (Not exposed to direct match): 84% E value - 8 x 10 ^ -22 Druggable Target (see databases for this): Yes Current Inhibitors: None, http: / / www.bindingdb.org/bind/ByMonomersTarget.jsp Expression Information: Purification Method: PyMol or other: Homology model with M. tuberculosis: Blastp gives 47% positives and 83 % query query. * Amino Acid Sequence:
Primers LmPTP-for imc TACTTCCAATCC ATG AAGAACTGGGTTAAAGTT LmPTP-rev imc AAAAGGCATACCTGTACTAA CAGTAAAGGTGGATA
Primers - C. Burns - Underlined is partial nucleotide sequence - middle black are start and stop codons
Forward - TACTTCCAATCCATGACCCTCGTTCTGCGTTAT
Reverse - TATCCACCTTTACTGTTAGTGTTCGAGGTCCGC ( link to DNA Works output text file -that should be saved in your Google Docs folder after you did the primary design protocol) - Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to. https://docs.google.com/document/d/1zCGuvqj1KwDp46nAn3SRRWRO2oCQd2O1F9vGN3blrsI/edit
Target (protein / gene name): Protein Tyrosine Phosphatase - lmo1800
* NCBI Gene # or RefSeq #: Project: 61583 , NC_003210.1, Gene ID: 985934
* Protein ID (NP or XP #) or Wolbachia #: NP_465325.1 (use De La O's Oligos for ordering - Dr. B 090313)
* Organism: Listeria monocytogenes
Etiologic Risk Group (see link below): Risk Group 2 - Bacterial agents including Chlaymidia
* Background / Disease Information: The Most Deadly Born food pathogen. Induces muscle aches, fever and diarrhea. Main route of infection is through uncooked and unwashed food.
Essentiality of this protein: Protein required to induce host to engulf bacteria. Upon entry to the cytosol bacteria can reproduce.
http://www.ncbi.nlm.nih.gov/pubmed/21444667
Complex of enzymes: No
* EC #: 3.1.3.48
Link to BRENDA EC # page: http://brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.48
Enzyme Assay information (spherophophometrics, coupled assay, reagents):
- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/search?interface=All&term=3.1.3.48&lang=en®ion=US&focuseddocuments&N = 0 + 220003049 + 219853269 + 219853286 & mode = match% 20partialmax ?????
- link to paper that contains information Assay:
http://www.brenda-enzymes.org/literature/lit.php4?e=3.1.3.48&r=663621
- List of cost and QUANTITY substrate reagents and Supplier
https://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=BAA-679D-5&Template=Bacteria
Structure Available (PDB or Homology model) http://www.rcsb.org/pdb /explore/explore.do?structureId=1YWF - QUERY Coverage (Not exposed to direct match): 84% E value - 8 x 10 ^ -22 Druggable Target (see databases for this): Yes Current Inhibitors: None, http: / / www.bindingdb.org/bind/ByMonomersTarget.jsp Expression Information: Purification Method: PyMol or other: Homology model with M. tuberculosis: Blastp gives 47% positives and 83 % query query. * Amino Acid Sequence:
http://www.rcsb.org/pdb/explore/explore.do?structureId=1YWF
> gi | 16803840 | ref | NP_465325.1 | lmo1800 hypothetical protein [Listeria monocytogenes EGD-e]
MKNWVKVTGAGVLSATLLLGGCGAQSEEKAEANVKTEQTLKPGSQIKLEGAVNVRDLGGYKTTDGLTIKPHKLIRSAEL
ANLSDSDKKKLVNTYDLSHIVDFRTSSEVATKPDPKLTDVDYTHDSVMKDNGTSTSTQDLTASLAKMDNPETFLINANKS
FITDETSIQAYKDFFDILLANQDGSVLWHCTAGKDRAGFGTALVLSALGVDKNTVIDDYMLSNKYRADENKKAIEAVAAKT
DNKKVIDGMTAVMEVRESYINAAFDEINAKYGSMDNFLKEKLGLTDAKKEQLKKAYLY
Molecular Weight of protein in your kiloDaltons using the Expasy ProtParam website
Molar Extinction coefficient of your protein at 280 nm wavelength:
* CDS Gene sequence:
Partial
> gi | 16802048: c1873620- 1872724 Listeria monocytogenes EGD-e, complete genomeATGAAAAATTGGGTAAAAGTAACAGGAGCAGGGGTACTAAGCGCGACTTTACTATTAGGTGGATGCGGGG CGCAGTCAGAAGAAAAAGCAGAAGCAAATGTTAAAACCGAGCAAACACTCAAACCAGGAAGCCAAATTAA ATTAGAAGGCGCTGTAAATGTCCGGGACTTAGGCGGATACAAAACAACGGATGGACTAACCATTAAGCCA CATAAACTCATTAGAAGTGCCGAACTCGCTAACTTAAGTGATTCGGATAAAAAGAAACTCGTAAATACAT ATGATTTGTCTCATATAGTTGATTTCCGAACAAGTTCAGAAGTCGCAACGAAACCAGATCCGAAACTCAC AGATGTAGACTATACGCACGATTCTGTGATGAAAGATAATGGAACATCTACAAGCACACAAGATTTAACT GCTAGCCTAGCCAAAATGGATAATCCAGAGACATTTCTCATTAATGCTAATAAGAGCTTTATTACAGATG AAACTTCGATACAAGCCTATAAAGATTTTTTTGATATACTACTAGCAAACCAAGATGGTTCTGTTCTTTG GCACTGTACAGCTGGAAAAGACCGAGCTGGATTTGGAACCGCCCTCGTTCTTTCAGCGTTAGGTGTGGAT AAAAACACGGTCATTGACGATTATATGCTGTCCAATAAATATCGTGCTGACGAAAATAAAAAAGCAATTG AAGCCGTTGCAGCAAAAACAGATAATAAAAAAGTGATTGATGGAATGACAGCCGTAATGGAAGTTCGTGA ATCTTATATCAATGCAGCCTTCGATGAAATTAATGCAAAATATGGTTCGATGGACAACTTTTTAAAAGAAAAACTCGGACTAACCGATGCTAATAACAACAACTAAAAAAAGCATATCTTTATTAA
* GC% Content for gene: 37.9%
* CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol:
ATGAAAAACTGGGTTAAAGTTACCGGTGCGGGCGTTCTCTCTGCTACTCTGCTGCTCGGT GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCAAACGTCAAGACCGAACAGACCCTG AAGCCGGGTAGCCAGATCAAACTGGAAGGTGCCGTTAATGTTCGCGACCTGGGTGGTTAC AAGACTACCGATGGTCTGACCATCAAACCGCACAAACTGATCCGCTCTGCGGAACTCGCA AACCTCTCTGACTCTGACAAAAAGAAGCTGGTTAACACCTACGACCTGTCTCACATCGTT GACTTCCGCACGTCTTCTGAAGTTGCGACCAAACCGGACCCGAAACTGACCGACGTGGAC TACACCCACGACTCTGTTATGAAAGACAACGGTACGTCTACCTCTACCCAGGACCTGACG GCGTCTCTGGCCAAGATGGACAACCCGGAAACCTTCCTGATTAACGCGAATAAATCCTTC ATTACCGACGAGACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTGGCGAAC CAGGACGGTTCCGTTCTGTGGCACTGCACCGCCGGTAAAGACCGTGCCGGTTTCGGCACC GCGCTGGTTCTGTCTGCGCTGGGTGTTGATAAAAACACCGTTATCGACGACTACATGCTG TCTAACAAATACCGTGCTGACGAAAATAAGAAAGCGATCGAAGCGGTAGCGGCGAAAACC GACAACAAAAAAGTGATCGACGGTATGACGGCAGTTATGGAAGTTCGTGAATCTTACATC AACGCGGCGTTCGATGAGATCAATGCGAAATATGGTTCTATGGATAACTTCCTCAAAGAAAAGCTGGGCCTCACTGATGCGAAGAAAGAGCAGCTGAAAAAAGCATACCTGTACTAA
*% GC content for Gene (codon optimized): 50.9%
primer design results for pNIC-Bsa4 cloning (list of all of your seqeunces ~ 40 nt long primers)
Oligos sequence
Lmon_PTP_1 || ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCT ||
|| Lmon_PTP_2 || ACGTTCGCTTCCGCTTTTCCTCAGACTGCGCACCACAACCACCCAGCAGCAGGGTCGCG ||
|| Lmon_PTP_3 || WHERE YOU SHOULD BE A BETWEEN YOUCCCCECCAACCGGTCTCTCAAATCAAACT ||
|| Lmon_PTP_4 || TTATAACCGCCGAGATCGCGCACGTTAACCGCACCTTCCAGTTTGATTTGAGAACCCGGT ||
|| Lmon_PTP_5 || GCGATCTCGGCGGTTATAAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCC ||
|| Lmon_PTP_6 || TCTTATCAGAGTCAGACAGATTCGCCAGTTCCGCAGAACGGATCAGTTTGGGTTTGA ||
|| Lmon_PTP_7 || CGAATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCG ||
|| Lmon_PTP_8 || TCCGGTTTGGTCGCAACTTCAGAAGAGGTGCGAAAGTCAACGATGTGAGACAGGTCGTAG ||
|| Lmon_PTP_9 || GTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGATTACACCCACGACTCTGTTATG ||
|| Lmon_PTP_10 || GGTCAGGTCTTGGGTGGAGGTAGAGGTACCGTTGTCTTTCATAACAGAGTCGTGGGTGTA ||
|| Lmon_PTP_11 || TCCACCCAAGACCTGACCGCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATC ||
|| Lmon_PTP_12 || TGGATAGAGGTTTCATCGGTGATAAAAGATTTGTTCGCATTGATCAGGAAGGTTTCCGGG ||
|| Lmon_PTP_13 || CACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAACCA ||
|| Lmon_PTP_14 || CGGTCTTTACCCGCAGTGCAGTGCCACAGGACAGAGCCGTCTTGGTTTGCGAGCAGGATG ||
|| Lmon_PTP_15 || CACTGCGGTAAAGACCGTGCTGGTTTCGGCACCGCGCTGGTCTCTCTGCACTCGGTGT ||
|| Lmon_PTP_16 || TGTTAGACAGCATGTAGTCGTCAATAACGGTGTTTTTGTCCACACCGAGTGCAGAGAGAA ||
|| Lmon_PTP_17 || ACGACTACATGCTGTCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGG ||
|| Lmon_PTP_18 || GTCATACCGTCGATAACTTTCTTGTTGTCGGTTTTCGCCGCAACCGTTTCGATCGCCTTT ||
|| Lmon_PTP_19 || CAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATTAACGC ||
|| Lmon_PTP_20 || ATAGAACCGTATTTTGCGTTGATTTCGTCGAACGCCGCGTTAATGTAAGATTCACGAACT ||
|| Lmon_PTP_21 || ATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAGAAACTGGGTCTGACGGAC ||
|| Lmon_PTP_22 || TTAGTACAGGTACGCTTTCTTCAGCTGTTCTTTTTCTCGCGTCCGTCAGACCCAGTTTC ||
Primers
LmPTP-for imc TACTTCCAATCC ATG AAGAACTGGGTTAAAGTT
LmPTP-rev imc AAAAGGCATACCTGTACTAA CAGTAAAGGTGGATA
Primers - C. Burns - Underlined is partial nucleotide sequence - middle black are start and stop codons
Forward - TACTTCCAATCCATGACCCTCGTTCTGCGTTAT
Reverse - TATCCACCTTTACTGTTAGTGTTCGAGGTCCGC
( link to DNA Works output text file -that should be saved in your Google Docs folder after you did the primary design protocol)
- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
https://docs.google.com/document/d/1zCGuvqj1KwDp46nAn3SRRWRO2oCQd2O1F9vGN3blrsI/edit
TMpred Output: